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        <title>Optical Calibration Overview</title>
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        <h1 align="center">Optical Calibration</h1>
		<p>Also see <a href="generating_an_ocf.html">Generating an OCF</a> and
		<a href="run_specific_ocf.html">Applying a Run-Specific OCF</a></p>
		<p>As described below, the LRE Analyzer relies on an &quot;optical 
		calibration factor&quot; or OCF for establishing absolute scale. When a new 
		experiment database is first created, the OCF is set to zero so that 
		target quantity cannot be determined:</p>
		<p align="center">
		<img border="0" src="images/ocf1.gif" width="425" height="216"></p>
		<p align="left">It is thus necessary to manually enter a value into the 
		OCF display box followed by return:</p>
		<p align="center">
		<img border="0" src="images/ocf2.gif" width="424" height="215"></p>
		<p align="left">In this case, the OCF was determined using lambda gDNA 
		as a quantitative standard (see the calibration demonstration database), 
		the data from which was processed within the
		<a href="../explorer_panel/calibration_database_window.html">calibration 
		database window</a>. However, the <a href="generating_an_ocf.html">Generating an OCF</a> 
		section describes alternative methods which can be used.</p>
		<p align="left"><b>Optical Calibration Overview<br>
		</b>One of the distinctive aspects of LRE quantification is that target 
		quantities are determined in fluorescence units (F<sub>0</sub>). 
		This provides&nbsp;the opportunity to apply an approach used by a 
		conventional fluorescence assay for quantifying DNA, in which sample 
		fluorescence is converted into DNA mass, based on the fluorescence 
		generated by a known quantity of a DNA standard, such as 
		lambda gDNA. Furthermore, if the sample is composed of a homogenous 
		mixture of DNA molecules, such as is the case for amplicon DNA within a 
		PCR reaction, the number of DNA molecules within the sample 
		can be determined if the size is known.</p>
		<p align="left">Absolute quantification can thus be achieved by using an 
		optical calibration factor to convert target quantity from fluorescence 
		units&nbsp; into DNA mass , followed by conversion into the number of 
		the number of DNA molecules based on amplicon size: </p>
		<p align="center">
		<img border="0" src="images/fo_conversion.gif" width="325" height="59"></p>
		<p align="left">Optical calibration is accomplished by correlated 
		fluorescence units to DNA mass, determined by amplifying a known quantity of DNA, 
		such as lambda gDNA. The resulting F<sub>0</sub> value is then used to 
		generate an optical calibration factor (OCF), expressed as 
		fluorescence units per nanogram of dsDNA:</p>
		<p align="center">
		<img border="0" src="images/ocf_equation.gif" width="212" height="71"></p>
		<p align="left">This then allows target quantity expressed in 
		fluorescence units (F<sub>0</sub>) to be converted in to DNA mass (M<sub>0</sub>), 
		which is equivalent to the mass of the amplicon region within the 
		target.:</p>
		<p align="center">
		<img border="0" src="images/mo_equation.gif" width="134" height="70"></p>
		<p align="left">The number of target molecules (N<sub>0</sub>) can 
		then be determined by converting DNA mass (M<sub>0</sub>) into the number of DNA 
		molecules, based on the amplicon size (A<sub>S</sub>), the number of 
		base pairs per ng of dsDNA, and the strandedness of the target:</p>
		<p align="center">
		<img border="0" src="images/no_equations.gif" width="456" height="187"></p>
		<p align="left">Note however, that this process assumes that 
		fluorescence accurately reflects amplicon DNA mass and is independent 
		amplicon size and GC content. Although extensive testing has shown that 
		SYBR Green I fulfills these requirements, other detection chemistries 
		may not.</p>
		<p align="left">Another important qualification is that the lambda gDNA 
		must be amplified using identical conditions to that used for sample 
		amplification. </p>
		<p>As described in the <a href="../quality_control/quality_control.html">quality control</a> 
		section, optical calibration also provides a number of quality control 
		capabilities that includes monitoring of run-to-run performance. The 
		universal nature of lambda gDNA also makes it amenable to developing 
		performance benchmarks that allow, for example, assessing 
		performance differences between different instruments and/or enzyme 
		formulation. </p>
    	<p>Note that a detailed description of optical calibration is 
		provided in the second video of the LRE Overview available on the LRE qPCR web site (<font color="#FF0000"><span style="text-decoration: none">sites.google.com/site/lreqpcr</span></font>). </p>
		<p>See also:<br>
		<a href="generating_an_ocf.html">Generating an OCF</a><br>
		<a href="run_specific_ocf.html">Applying a Run-Specific OCF</a></p>
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